Method for purifying protein

ABSTRACT

The present invention provides a method for purifying a protein to remove impurities from a mixture liquid containing a desired protein and the impurities, comprising performing filtration using a porous membrane having a graft chain on a pore surface and an anion-exchange group fixed to the graft chain.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.12/681,189, which is a National Stage of International PatentApplication No. PCT/JP2008/067540, filed Sep. 26, 2008, and claimspriority to Japanese Application No. 2007-279406, filed Oct. 26, 2007.The disclosures of application Ser. Nos. 12/681,189 andPCT/JP2008/067540 are expressly incorporated by reference herein intheir entireties.

TECHNICAL FIELD

The present invention relates to a method for purifying a protein.Specifically, the present invention relates to a method for easilyremoving impurities from a mixture liquid containing a desired proteinand the impurities, represented by an animal cell culture andefficiently purifying the desired protein.

BACKGROUND ART

In recent years, the practical large-scale purification of a protein hasrepresented an important challenge in the biotechnology-based industry.Particularly in the field of medicine, demand for antibody drugs israpidly increasing; there is a strong desire to establish technologiescapable of efficiently producing and purifying large amounts of aprotein.

A protein is generally produced by cell culture using a cell linederived from an animal. For practical use of a desired protein(hereinafter sometimes referred to simply as “a target protein”),particularly an antibody drug used as a medicine, it is necessary toremove cell debris and the like providing turbid components and acell-derived dissolved protein and the like providing non-turbidcomponents from a cell culture, followed by purification to such adegree as to give a composition sufficient for therapeutic applicationin humans.

A typical operation of purifying a target protein from a cell culturecomprises first subjecting the cell culture to centrifugation toprecipitate and remove turbid components; then, removing cell debrishaving a size of about 1 μm or less incapable of being completelyremoved by the centrifugation among the turbid components by means ofsize filtering using a microfiltration membrane; and further subjectingthe filtrate to sterilization filtration using a filtration membranehaving a maximum pore size of 0.22 μm or less for sterilization toprovide a clear solution of the target protein (a harvest step). Whenthe clear solution containing the target protein is obtained, the targetprotein is subsequently separated and purified using a purificationprocess employing a combination of a plurality of chromatographictechniques including affinity chromatography (a downstream step).Impurity proteins produced from cells in the culture and dissolved inthe culture are removed in the downstream step. In such a conventionalmethod by which the target protein and the like are purified from thecell culture, the concentration of the target protein in the culture istypically 1 g/L or less, and the concentration of cell debris, dissolvedimpurity proteins and the like contained in the culture is alsocomparable to the concentration of the target protein. In theconcentration range, the purification process using the conventionalharvest and downstream steps is sufficiently useful in purifying thetarget protein.

However, a rapid increase in demand for antibody drugs allows theproduction of proteins providing the antibody drugs to belarge-scale-oriented; fast-paced advances in culture techniques haverecently increased the concentration of a target protein in a cellculture, which is about to reach as high as 10 g/L. Such fast-pacedadvances in culture techniques simultaneously mean the result as whichimpurity proteins also increase, causing the prediction of substantialloads applied to purification using a conventional protein purificationprocess.

Hence, as a technique for purifying large amounts of a protein, forexample, Patent Documents 1 and 2 each disclose a protein adsorptionmembrane to which protein adsorption capability is imparted byintroducing ion-exchange groups into a porous membrane, which can alsobe purchased.

As a usage example of a protein adsorption membrane, Patent Document 3also discloses a method for separating albumin from a lymph fluid usingtwo types of protein adsorption membranes, i.e. a porous cellulosemembrane into which anion-exchange groups are introduced and a porouscellulose membrane into which cation-exchange groups are introduced.

Further, Patent Document 4 discloses a method for separating nucleicacid and endotoxin using a porous cellulose membrane into whichanion-exchange groups are introduced.

Sill further, Patent Documents 5 and 6 disclose protein adsorptionmembranes in which cation-exchange groups and anion-exchange groups,respectively are introduced into porous polyether sulfone membranes.

Non-Patent Document 1 discloses a method for separating nucleic acid andmonoclonal antibody using a porous membrane containing ion-exchangegroups.

-   Patent Document 1: U.S. Pat. No. 5,547,575-   Patent Document 2: U.S. Pat. No. 5,739,316-   Patent Document 3: U.S. Pat. No. 6,001,947-   Patent Document 4: U.S. Pat. No. 6,235,892-   Patent Document 5: U.S. Pat. No. 6,783,937-   Patent Document 6: U.S. Pat. No. 6,780,327-   Non-Patent Document 1: Bioseparation 8:281-291, 1999

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

However, aggregated impurities are often observed to be contained in arecovered solution when a cell culture containing a high concentrationof a target protein is subjected to centrifugation, microfiltration andsterilization filtration to make a clear fluid, which is then applied toa protein A affinity column as an affinity chromatography column toselectively adsorb the target protein, followed by eluting and recoverthe protein with an acidic eluent. With increasing number of repeateduses of the protein A affinity column, the amount of the aggregatedimpurities tends to be increased while the amount of the recoveredtarget protein being decreased.

A larger amount of impurity proteins increases loads in the affinitychromatography step as described above, and not only requires a longertime of the purification step owing to the washing of the column and thelike, but also results in shorter life of column beads used in theaffinity chromatography, particularly the life of the protein A affinitycolumn. It is undesirable for the protein A affinity column to haveshorter life because the column is expensive.

In each of the methods disclosed in Patent Documents 1 to 4, theseparation operation is performed after passing a stock solution used inthe separation through a microfiltration membrane in advance to removeparticulate impurities. This is because the protein adsorption membranecannot remove particulate impurities since the protein adsorptionmembrane has a maximum pore size of as large as 3 μm to 5 μm.

Also in each of the methods disclosed in Patent Documents 5 and 6, theprotein adsorption membrane cannot remove fine particulate impuritiessince it has a maximum pore size of 0.8 μm to 1.0 μm.

In addition, a cell culture generally contains a salt; when anion-exchange membrane is used as disclosed in Non-Patent Document 1,impurity proteins cannot be practically removed from the cell culturebecause the amount of adsorption of proteins from a solution containing0.1 M or more of the salt is markedly decreased.

As described above, the existing protein adsorption membranes havemaximum pore sizes of about 0.8 μm or more and decreased amounts ofadsorption in the presence of a salt. Thus it is not assumed that theadsorption membranes are used to simultaneously perform clarificationintended to remove fine insoluble matter such as cell debris andadsorption of dissolved impurity proteins; the adsorption membranes arenot suitable for the purpose.

Each of the protein adsorption membranes has a dynamic adsorption volumeof 30 mg/mL or less per membrane volume even under conditions in whichno salt is present in the solution; thus, a large amount of the proteinadsorption membrane will be required to effectively adsorb and removeimpurity proteins from the cell culture. In addition, each of theseexisting protein adsorption membranes is in the form of a flat membraneand therefore a large amount of the protein adsorption membrane cannotbe housed in a compact size. As such, a large amount of the proteinadsorption membrane housed in a large-size container will be requiredand thus cannot be said to be practical. The existing protein adsorptionmembranes having anion-exchange groups are more unsuitable for practicaluse because their amount of adsorption is markedly decreased when a saltis present in the solution.

In view of such circumstances, an object of the present invention is toprovide a method for easily removing impurities from a mixture liquidcontaining a target protein and the impurities, represented by an animalcell culture and efficiently purifying the target protein.

Means for Solving the Problems

As the result of intensive studies for solving the above-describedproblems, the present inventors have found that the use of a porousmembrane having graft chains on the pore surface and anion-exchangegroups fixed to the graft chains is surprisingly effective to solve theabove problems.

Thus, the present invention provides a method for purifying a proteinand a porous hollow fiber membrane as described below.

[1] A method for purifying a protein to remove impurities from a mixtureliquid containing a desired protein and the impurities, comprising thestep of:

performing filtration using a porous membrane having a graft chain on apore surface and an anion-exchange group fixed to the graft chain.

[2] The method for purifying the protein according to item [1] above,wherein the desired protein is one selected from the group consisting ofmonoclonal antibodies, polyclonal antibodies, humanized antibodies,human antibodies and immunoglobulins.[3] The method for purifying the protein according to item [1] or [2]above, wherein the impurities are at least one selected from the groupconsisting of a non-turbid component and a turbid component dispersed inthe mixture liquid.[4] The method for purifying the protein according to item [3] above,wherein the non-turbid component is at least one selected from the groupconsisting of impurity proteins, HCP, DNA, viruses, endotoxins,proteases and bacteria dissolved in the mixture liquid.[5] The method for purifying the protein according to item [3] or [4]above, wherein the turbid component dispersed in the mixture liquid isat least one selected from the group consisting of cells and celldebris.[6] The method for purifying the protein according to any one of items[1] to [5] above, wherein a salt concentration of the mixture liquid isfrom 0.01 M to 0.5 M (both inclusive).[7] The method for purifying the protein according to any one of items[1] to [5] above, wherein a salt concentration of the mixture liquid isfrom 0.1 M to 0.3 M (both inclusive).[8] The method for purifying the protein according to any one of items[1] to [7] above, wherein:

a base material of the porous membrane is polyethylene or polyvinylidenefluoride,

the graft chain is a polymer of glycidyl methacrylate and has a graftrate of from 10% to 250% (both inclusive), and

the graft chain has 70% or more of epoxy groups replaced with theanion-exchange groups.

[9] The method for purifying the protein according to item [8] above,wherein the graft rate is from 10% to 150% (both inclusive).[10] The method for purifying the protein according to item [8] above,wherein the graft rate is from 10% to 90% (both inclusive).[11] The method for purifying the protein according to item [8] above,wherein the graft rate is from 30% to 60% (both inclusive).[12] The method for purifying the protein according to any one of items[1] to [11] above, wherein the anion-exchange group is a diethylaminogroup and/or a trimethylamino group.[13] The method for purifying the protein according to any one of items[1] to [12] above, wherein the anion-exchange group is a diethylaminogroup.[14] The method for purifying the protein according to any one of items[1] to [13] above, wherein the porous membrane has a maximum pore sizeof from 0.1 μm to 0.8 μm (both inclusive).[15] The method for purifying the protein according to any one of items[1] to [14], wherein the mixture liquid is filtered using the porousmembrane to remove one or more impurities comprising a non-turbidcomponent.[16] The method for purifying the protein according to any one of items[1] to [15], wherein the mixture liquid is an animal cell culture.[17] The method for purifying the protein according to any one of items[1] to [16], wherein the porous membrane is a porous hollow fibermembrane.[18] A porous hollow fiber membrane used in the method for purifying theprotein according to item [17] above.[19] A module comprising the porous hollow fiber membrane according toitem [18] above.[20] A porous hollow fiber membrane having a graft chain on a poresurface and an anion-exchange group fixed to the graft chain, wherein:

a base material of the porous hollow fiber membrane is polyethylene orpolyvinylidene fluoride,

the graft chain is polymers of glycidyl methacrylate and has a graftrate of from 10% to 250% (both inclusive), and

the graft chain has 70% or more of epoxy groups replaced with theanion-exchange group.

[21] The porous hollow fiber membrane according to item [20] above,wherein the graft rate is from 10% to 150% (both inclusive).[22] The porous hollow fiber membrane according to item [20] above,wherein the graft rate is from 10% to 90% (both inclusive).[23] The porous hollow fiber membrane according to item [20] above,wherein the graft rate is from 30% to 60% (both inclusive).[24] The porous hollow fiber membrane according to any one of items [20]to [23] above, wherein the porous membrane has a maximum pore size offrom 0.1 μm to 0.8 μm (both inclusive).[25] A module comprising the porous hollow fiber membrane according toany one of items [20] to [24] above.[26] The method for purifying a protein according to any one of items[1] to [17] above, further comprising the step of performingpurification using affinity chromatography.

Advantages of the Invention

In accordance with the method for purifying a protein according to thepresent invention, the clarification of a cell culture before anaffinity chromatography step, carried out by the three steps ofcentrifugation, microfiltration and sterilization filtration can beeasily performed through filtration using a porous membrane having graftchains and anion-exchange groups fixed to the graft chains.

In addition, removal of dissolved impurity proteins incapable of beingremoved by conventional methods can be made possible by filtration withthe porous membrane having the fixed anion-exchange groups. Further, itis made possible to greatly reduce loads in the affinity chromatographystep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results of the SDS-PAGE analysis of straightlypassed-through and adsorbed components in the filtration of a mixtureliquid of BSA and γ-globulin using an anion-exchange hollow fibermembrane module in Example 3;

FIG. 2 shows results of the SDS-PAGE analysis of the purification of aγ-globulin-containing cell culture by filtration using variousanion-exchange membranes in Examples 4 to 6 and Comparative Example 1;

FIG. 3 illustrates a schematic drawing of an estimation apparatus forperforming clarification, impurity protein removal and sterilization bya single step using an anion-exchange hollow fiber membrane module inExample 10;

FIG. 4 shows results of the SDS-PAGE analysis of straightlypassed-through and adsorbed components in the filtration using ananion-exchange hollow fiber membrane module in Example 10; and

FIG. 5 illustrates a schematic drawing of an estimation apparatus forperforming impurity protein removal and sterilization by 2 steps usingan anion-exchange hollow fiber membrane module after clarification witha microfiltration hollow fiber membrane module in Example 11.

DESCRIPTION OF SYMBOLS

-   1 Cell culture tank-   2 Perister pump-   3 Pressure indicator (at entry side of module)-   4 Anion-exchange hollow fiber membrane module-   5 Pressure indicator (at exit side of module)-   6 Flow control cock-   7 Microfiltration hollow fiber membrane module for clarification

BEST MODE FOR CARRYING OUT THE INVENTION

A best mode for carrying out the present invention (hereinafter referredto as “the present embodiment”) is described below in detail. Note thatthe present invention is not intended to be limited to the followingembodiment, and various modifications can be made within the scope ofthe gist of the invention.

The method for purifying a protein according to the present embodimentis a method for purifying a protein to remove impurities from a mixtureliquid represented by an animal cell culture, containing a desiredprotein useful as a medicine and the impurities, comprising the step ofperforming filtration using a porous membrane having a graft chain on apore surface and an anion-exchange group fixed to the graft chain.

The method for purifying a protein according to the present embodimentis a method for purifying a protein which can easily remove impuritiescontained in a mixture liquid to provide a clear solution of a targetprotein.

Preferably, the method for purifying the protein according to thepresent embodiment further comprises the step of purifying the cleartarget protein solution obtained by the filtration step using affinitychromatography.

Examples of the mixture liquid containing a desired protein andimpurities can include, but not limited to, an animal cell culture.

The animal cell culture is not particularly limited provided that it isa culture containing a target protein; however, examples thereof caninclude a culture containing a recombinant protein obtained by cellculture from host cells including Chinese hamster ovary (CHO) cells.

Examples of the target protein can include antibodies used as medicine;examples thereof can include monoclonal antibodies, polyclonalantibodies, humanized antibodies, human antibodies and immunoglobulins.

Examples of the impurities can include a turbid component dispersed in amixture liquid and a non-turbid component.

Examples of the turbid and non-turbid components can include impuritiesother than a target protein contained in the culture obtained byperforming animal cell culture in order to produce the desired protein.

Examples of the turbid component dispersed in a mixture liquid caninclude cells and cell debris; examples of the non-turbid component caninclude impurity proteins, host cell proteins (HCP), nucleic acids(DNA), viruses, endotoxins, proteases, and bacteria dissolved in themixture liquid.

The method for purifying the protein according to the present embodimentpreferably involves filtering the mixture liquid using the porousmembrane to remove one or more impurities including the non-turbidcomponent.

The non-turbid component can be removed by adsorption to the porousmembrane. Preferably, the turbid component is also removed in removingone or more impurities including the non-turbid component.

The isopotential point (pI) of an antibody as a target protein rangesfrom about 6 to 8. A typical CHO animal cell culture has a pH rangingfrom roughly 7 to 8 and contains about 1% by mass (about 0.17 M) of asalt. The pH and salt concentration can vary depending on the type ofthe animal cell culture.

In an aqueous solution, the amino and carboxyl groups of the protein arepresent in ionized states: —NH₃ ⁺ and —COO⁻, respectively and the totalcharge of the protein in the aqueous solution depends on the pH of theculture. The total charge of the protein is zero at the pH of theisoelectric point (pI). The protein is made in a negatively chargedstate at a pH of more than pI, and in a positively charged state at a pHof less than pI.

Because an animal cell culture has a pH of from 7 to 8, a target proteinis present in the culture in such a state that the protein has a totalcharge of nearly zero or is slightly positively charged; thus, thetarget protein is not substantially adsorbed by an anion-exchange group.

In contrast, the impurity proteins, host cell proteins (HCP), nucleicacids (DNA), endotoxins and the like dissolved in a mixture liquid arepresent in a negatively charged state in an animal cell culture evenwhen dissolved in the culture because most of them have a pH of 6 orlower. The impurity proteins and the like dissolved in the mixtureliquid have the property of being adsorbed by an anion-exchange groupunder conditions in which a salt is absent.

However, an animal cell culture typically contains a salt on the orderof about 1% by mass (0.17 M) in practice, allowing few impurity proteinsand the like to be adsorbed by an anion-exchange group even at a pI of 6or lower. Thus, impurities cannot be directly removed from an animalcell culture even by use of a conventional adsorption membrane havinganion-exchange groups or anion-exchange chromatography.

The method for purifying the protein according to the present embodimentcan be used to purify even an animal cell culture having a saltconcentration of from 0.01 M to 0.5 M (both inclusive).

According to the present embodiment, the salt concentration of theanimal cell solution capable of being preferably purified is from 0.1 Mto 0.3 M (both inclusive).

According to the present embodiment, when an animal cell culture isfiltered using the porous membrane having the graft chains on the poresurface and the anion-exchange groups fixed to the graft chains, it hassurprisingly been found that the impurity proteins, host cell proteins(HCP), nucleic acids (DNA), endotoxins and the like dissolved in themixture liquid are adsorbed by the anion-exchange groups of the porousmembrane despite that a salt is contained in the cell culture.

The porous membrane having graft chains on the pore surface andanion-exchange groups fixed to the graft chains is used in thefiltration process to enable that adsorption of the impurity proteins,host cell proteins, nucleic acids, endotoxins and the like dissolved ina mixture liquid from an animal cell culture, which is not typicallyachieved. Although the detailed reason is unclear, the following isspeculated.

In a common anion-exchange membrane and anion-exchange chromatography,anion-exchange groups are generally fixed to the surface of porousmembrane pores or a resin and its pores. A protein is adsorbed bysurface anion-exchange groups. The anion-exchange groups are fixed onlyto the surface of porous membrane pores or a resin and its pores andthereby rest two-dimensionally. As such, the protein is adsorbed in apoint-contact manner on the surface of porous membrane pores or a resinand its pores; thus, the anion-exchange groups capable of beingresponsible for the adsorption are small in number. Thus, the presenceof a salt in the solution significantly reduces the adsorption of theproteins.

This is the common concept about an anion-exchange membrane; based onthis principle, the use of a salt solution is adopted as a conventionalmethod in eluting adsorbed proteins.

In other words, the anion-exchange membrane is thought to adsorb noprotein when a salt is present in the solution; this concept has beenditto for an anion-exchange membrane having graft chains.

In the porous membrane used in the present embodiment, anion-exchangegroups are fixed to graft chains present on the surface of pores.

The present inventors have found the fact that the use of the porousmembrane of the present embodiment, i.e. the anion-exchange membranehaving anion-exchange groups fixed to graft chains, does not cause asignificant reduction in the adsorption of a protein even in a solutioncontaining a salt.

The adsorption of a protein is two-dimensional for a conventional porousmembrane in which anion-exchange groups rest only on the surface ofpores or a resin, whereas the fixation of anion-exchange groups to graftchains allows the anion-exchange groups to be three-dimensionally(sterically) placed. The fixation of anion-exchange groups to graftchains is thought to increase the number of anion-exchange groupsresponsible for adsorption because graft chains adsorb onto a protein insuch a manner that the protein become entangled with the chains. Theincreased number of the anion-exchange groups involved in the adsorptioncauses little reduction in the adsorption of a protein even when a saltis present in the solution, which probably achieves the removal ofdissolved impurities by adsorption even from a practical cell culture.

The porous membrane having anion-exchange groups used in the presentembodiment refers to a porous membrane composed of a porous body havinggraft chains fixed to the surface of the porous body as a base materialand its pores and anion-exchange groups chemically or physically fixedto the graft chains.

The base material for the porous membrane is not particularly limited;however, it is preferably composed of a polyolefin polymer for retentionof mechanical properties.

Examples of the polyolefin polymer can include homopolymers of olefinssuch as ethylene, propylene, butylene and vinylidene fluoride,copolymers of two or more of the olefins, or copolymers of one or two ormore of olefins and perhalogenated olefins.

Examples of the perhalogenated olefin can include tetrafluoroethyleneand/or chlorotrifluoroethylene.

Among these base materials, preferred is polyethylene or polyvinylidenefluoride, more preferably polyethylene, in that they are raw materialsexcellent particularly in mechanical strength and providing a highadsorption volume.

Methods for introducing graft chains into the surface and pores of aporous membrane and further fixing anion-exchange groups to the graftchains include, for example, but not limited to, a method as disclosedin Japanese Patent Laid-Open No. 02-132132.

According to the present embodiment, the graft rate refers to the ratio(percentage) of the weight of the graft chains introduced into the basematerial before the fixation of anion-exchange groups to the graftchains, to the weight of the base material.

The graft rate is preferably from 10% to 250% (both inclusive), morepreferably from 10% to 150% (both inclusive), still more preferably from10% to 90% (both inclusive), yet more preferably from 30% to 60% (bothinclusive).

The graft rate of 10% or more makes the adsorption volume of a proteinsignificantly high and is practical.

The graft rate of 250% or less provides practical strength.

A salt solution is generally passed through the porous membrane ineluting the adsorbed protein, which is characterized by expanding themembrane volume; a higher graft rate results in a higher expansioncoefficient. The expansion coefficient of the membrane volume due to thepassage of the salt solution depends on the structures of the porousmembrane and graft chain; however, it is about 2% or less for the graftrate of 60%, about 3% or less for 90%, and about 5% or less for 150%. Agraft rate of 250% or less can make the expansion coefficient 10% orless; thus, a porous membrane having a graft rate of 250% or less ispractically preferable.

According to the present embodiment, the graft chain introduced into thebase material refers to a chain having a chemical structure which is notremoved even by washing with an organic solvent such asdimethylformamide (DMF) after the introduction reaction.

Examples of the graft chain can include polymers of glycidylmethacrylate, vinyl acetate and hydroxypropyl acetate; however,preferred is a polymer of glycidyl methacrylate or vinyl acetate, morepreferably a polymer of glycidyl methacrylate since anion-exchangegroups are easily introduced thereinto.

The anion-exchange group is not particularly limited provided that it isan anion-exchange group capable of adsorbing an impurity protein, DNA,HCP, a virus, an endotoxin or the like dissolved in the mixture liquid;examples thereof can include a diethylamino (DEA, Et₂N—) group, aquaternary ammonium (Q, R₃N⁺—) group, a quaternary aminoethyl (QAE,R₃N⁺—(CH₂)₂—) group, a diethylaminoethyl (DEAE, Et₂N—(CH₂)₂—) group, anda diethylaminopropyl (DEAP, Et₂N—(CH₂)₃—) group. R is not particularlylimited, and R's bonded to the same N may be the same or different; Rpreferably represents a hydrocarbon group such as an alkyl group, aphenyl group and an aralkyl group.

Examples of the quaternary ammonium group can include a trimethylaminogroup (a trimethylammonium group introduced into a graft chain, Me₃N⁺—).

Preferred are DEA and Q, more preferably DEA since these groups areeasily fixed chemically to the graft chain introduced into the porousmembrane and provide a high adsorption volume.

The anion-exchange group can be fixed to the graft chain by ring-openingthe epoxy group of the glycidyl methacrylate polymer forming the graftchain and adding an amine such as diethylamine and an ammonium salt suchas diethylammonium or trimethylammonium.

Seventy percent or more, preferably 75% or more, more preferably 80% ormore by mole fraction of the epoxy groups of the graft chain arepreferably replaced with anion-exchange groups. The amount of thereplacing anion-exchange groups being within the above range can make aporous membrane having a high dynamic adsorption volume.

The maximum pore size of the porous membrane is preferably from 0.1 μmto 0.8 μm (both inclusive), more preferably from 0.1 μm to 0.6 μm (bothinclusive), still more preferably from 0.2 μm to 0.5 μm (both inclusive)to cut a turbid component and bacteria and yet to obtain a high flux.

According to the present embodiment, the maximum pore size of the porousmembrane refers to a value as determined by a bubble point method asgiven in Examples.

The dynamic adsorption volume for proteins from a salt-free solution,which the porous membrane containing anion-exchange groups has ispreferably 30 mg/mL or more, more preferably 50 mg/mL or more, stillmore preferably 70 mg/mL or more to effectively reduce loads on anaffinity chromatography column in the purification step using affinitychromatography.

The dynamic adsorption volume when the buffer contains 0.1 mol/L of asalt is preferably 10 mg/mL or more, more preferably 20 mg/mL or more,still more preferably 30 mg/mL or more.

The amount of impurities adsorbed to an ion-exchange membrane isproportional to the volume of the ion-exchange membrane. Thus, thelarger dynamic adsorption volume of the ion-exchange membrane candecrease the size of a module used for purifying a protein.

According to the present embodiment, the dynamic adsorption volumerefers to the mass (in mg/mL) of a protein adsorbed by a porous membranebefore breakthrough per volume of the porous membrane. Bovine serumalbumin (BSA) is used as a model protein for estimating the adsorptionvolume; BSA dissolved in a 20 mM Tris-HCl (pH 8.0) buffer can be used toestimate the dynamic adsorption volume by a method as described in thefollowing Examples.

The form of the porous membrane is not particularly limited providedthat it is a porous body; examples thereof can include a flat membrane,nonwoven fabric, hollow fiber membrane, monolith, capillary, disk orcylinder form.

The porous membrane is preferably a hollow fiber membrane in view ofease of production, scalability, membrane packing properties in modularmolding, and the like.

According to the present embodiment, the porous hollow fiber membranehaving anion-exchange groups can be used in the harvest step before thedownstream step employing affinity chromatography to filter an animalcell culture containing a target protein to provide a clear solution ofthe target protein. The porous hollow fiber membrane havinganion-exchange groups can be used to remove impurity proteins, HCP, DNA,viruses and the like as non-turbid components, present in a dissolvedstate in the culture in addition to cells, cell debris and the likepresent as turbid components and yet remove bacteria to provide a cleartarget protein solution which is made free of impurities and sterilized.

For the purpose of obtaining a clear target protein solution from thecell culture, the porous hollow fiber membrane having anion-exchangegroups is preferably housed in a module.

The porous hollow fiber membrane module is a module housing a poroushollow fiber membrane composed of a porous hollow fiber in whichanion-exchange groups are chemically or physically fixed to the surfaceof the fiber and the surface of its pores. Using the porous hollow fibermembrane module, an animal cell culture can be passed therethrough toremove cells, cell debris and the like present as turbid components anddissolved impurity proteins, HCP, DNA, bacteria and the like present asnon-turbid components from the culture. The cells, cell debris and thelike present in the culture as turbid components can be removed by sizefiltering since they are large in size and thus cannot be passed throughthe pores of the porous hollow fiber. Impurity proteins and the likepresent dissolved in the culture can be removed by adsorption to theanion-exchange groups fixed to the surface of the porous hollow fiberand the surface of its pores.

According to the present embodiment, to remove impurities such as turbidand non-turbid components in an animal cell culture, a cross-flowfiltration method is preferably used which involves passing apressurized culture through the inside of a porous hollow fiber to passthe culture out to the outside of the porous hollow fiber.

The culture can be passed to the inside of the porous hollow fiber bycross-flow to inhibit the sedimentation of turbid material on the insidesurface in passing the culture out to the outside of the porous hollowfiber to suppress a considerable reduction in the flux.

In the cross-flow filtration, the linear velocity of the liquid inflowing through the porous hollow fiber is preferably 0.05 m/s to 5.0m/s, more preferably 0.1 m/s to 2.0 m/s.

According to the present embodiment, the linear velocity is the velocityof a liquid passing through the inner section of the porous hollowfiber, and is expressed by: (the volume of the liquid flowing throughthe porous hollow fiber per second)/(the inner sectional area of theporous hollow fiber).

The filtering liquid passage pressure from the inside of the poroushollow fiber to the outside thereof is preferably from 0.01 MPa to 0.5MPa, more preferably from 0.05 MPa to 0.2 MPa.

According to the present embodiment, preferred methods for obtaining aclear solution of a target protein from an animal cell culture in ashort time also include a method which involves subjecting the animalcell culture only to clarification treatment and then performing afiltration step using the porous hollow fiber membrane havinganion-exchange groups. The animal cell culture can be subjected only toclarification treatment in advance before filtration using the poroushollow fiber membrane having anion-exchange groups to provide a highflux in the filtration step, enabling the filtration step employing theporous hollow fiber membrane to be performed by normal flow filtration.

The clarification treatment is preferably performed in advance becausethe filtration using the porous hollow fiber membrane havinganion-exchange groups removes only non-turbid components such asresidual cell debris, dissolved impurity proteins; HCP, DNA, andbacteria.

Non-limited examples of the clarification treatment of an animal cellculture can include a method using centrifugation, normal flowfiltration with a depth filter such as a diatomaceous earth filter, orcross-flow filtration with a microfiltration membrane.

The liquid subjected to clarification treatment can be directly fed tofiltration using the porous hollow fiber membrane having anion-exchangegroups; thus, the cross-flow filtration with a microfiltration membraneis preferable, and cross-flow filtration with a microfiltration hollowfiber membrane module is more preferable.

The maximum pore size of the porous hollow fiber in the microfiltrationhollow fiber membrane module used for clarification treatment ispreferably from 0.1 μm to 0.8 μm, more preferably from 0.2 μm to 0.6 μm.

A smaller maximum pore size provides no sufficient flux, and a largermaximum pore size tends to cause plugging in subsequent normal flowfiltration through the hollow fiber membrane module comprising theporous hollow fibers having anion-exchange groups according to thepresent embodiment, resulting in increased passage pressure.

In performing the clarification treatment by cross-flow filtration usingthe microfiltration hollow fiber membrane module, the linear velocity ofthe animal cell culture when flowing on the inner surface of the poroushollow fiber is preferably from 0.05 m/s to 5.0 m/s, more preferablyfrom 0.1 m/s to 2.0 m/s.

The filtering liquid passage pressure from the inside of the poroushollow fiber to the outside thereof is preferably from 0.01 MPa to 0.5MPa, more preferably from 0.05 MPa to 0.2 MPa.

Preferably, the method for purifying the protein according to thepresent embodiment further comprises the step of performing purificationusing affinity chromatography.

The purification using affinity chromatography can be carried out by aheretofore known method and can be performed using a protein A affinitycolumn.

In this process, the clarified solution containing a target proteinobtained by filtration using the porous membrane having anion-exchangegroups is first applied to the protein A affinity column for theselective adsorption of the target protein.

The dissolved impurity proteins contained in the clarified solution areremoved by outflow without adsorption to the protein A affinity column.Next, the column is washed with a buffer having the same pH as that ofthe clarified solution to remove impurities left in the column, and theadsorbed target protein can be then eluted and recovered using an acidiceluent to provide a solution of a purified target protein from whichmost of the impurity proteins have been further removed.

Even with impurity proteins being dissolved in a solution to be appliedcontaining the target protein, the target protein can be purified usingthe protein A affinity column.

In the step of performing purification by affinity chromatography, alarge amount of dissolved impurity proteins results in the presence ofaggregated impurities in the recovered solution from the column, orreduces the life of the column because loads are applied to the column.Before application to the column, dissolved impurity proteins arepreferably removed as much as possible in advance using the poroushollow fiber membrane having anion-exchange groups; thus, it ispreferred to filter them using the porous membrane used in the presentembodiment.

Examples of the affinity chromatography column used in the presentembodiment can include columns having protein A, heparin, Con A, Red(Procion Red HE-3B), Blue (Cibacron Blue 3GA), lysine, arginine,benzamidine, and the like as ligands; when the target protein is anantibody, the protein A affinity chromatography column is often used.

The method for applying the clarified solution containing a targetprotein obtained by filtration using the porous membrane havinganion-exchange groups to the affinity chromatography column foradsorption can be carried out by a method which involves, for example,supplying the clarified solution to the column after equilibrating usinga pump or hydrostatic pressure.

The washing of the affinity chromatography column to which the targetprotein is adsorbed is not particularly limited provided that it uses abuffer having the same pH as that of the clarified solution containingthe target protein; examples of the buffer can include a buffer such asa sodium-phosphate buffer.

The washing with the buffer can be carried out by passing a bufferhaving a volume of on the order of 2 to 10 times the column volumethrough the column.

The recovery of the target protein from the affinity chromatographycolumn to which the target protein is adsorbed is not particularlylimited provided that it uses a buffer of an acidic solution as anelution buffer; examples of the buffer can include acidic solutions suchas a pH 3 to 4 citrate-NAOH buffer.

The recovery of the target protein using the acidic solution can becarried out by feeding the elution buffer at a volume of 2 to 10 timesthe column volume through the column.

The previously observed generation of aggregated impurities occurring inthe recovery of a protein with an acidic solution can be suppressed byfiltering an animal cell culture containing a target protein using theporous membrane having anion-exchange groups to provide a clear solutionof the target protein and then purifying the clear solution of thetarget protein by affinity chromatography to recover the target protein.A mixture liquid containing a target protein can be filtered using theporous membrane employed in the present embodiment to reduce loads onthe affinity column as well as to recover a highly purified targetprotein.

EXAMPLES

The present embodiment is more specifically described below based onExamples and Comparative Examples. However, the present embodiment isnot intended to be limited only to these Examples. Estimation andmeasurement methods used in the present embodiment are as follows.

(1) Bubble Point Method

The bubble point method was used to measure the maximum pore size of aporous hollow fiber. One end of a porous hollow fiber 8 cm in length wasblocked, and the other end was connected to a nitrogen gas supply linevia a pressure indicator. Nitrogen gas was supplied in the connectedstate to replace the inside of the line with nitrogen, followed byimmersing the porous hollow fiber in ethanol. At this time, theimmersion was carried out in a state only slightly pressurized withnitrogen to avoid backflow of ethanol into the line. The pressure ofnitrogen gas was slowly increased in a state in which the porous hollowfiber was immersed, and the pressure (p) at which bubbles of nitrogengas started to stably occur from the porous hollow fiber was recorded.

The maximum pore size of the porous hollow fiber was calculated by

d=Cγ/p  (I)

wherein d is the maximum pore size and γ is the surface tension of theinterface between ethanol and air.

Here, C is a constant. Because the immersion fluid is ethanol, C=0.632(kg/cm); the maximum pore size d (μm) was determined by substituting p(kg/cm²) into the above equation.

(2) Bacteria Challenge Test

A bacteria challenge test was carried out to determine the sterilizationperformance of minimodules as prepared in Examples 1, 2 and 6.

A 100 ppm sodium hypochlorite aqueous solution (200 mL) was fed to, andsimultaneously passed through, the inside of the module in the manner ofcross-flow for sterilization, and then extra pure water (500 mL) wassimilarly fed while passing therethrough for washing. Using Pseudomonasdimunuta as an indicator bacterium for a pore size of 0.22 μm, 200 mL ofan aqueous solution containing a concentration of 10⁶ cells/mL of theindicator bacteria was fed to, and simultaneously passed through, theinside thereof also in the manner of cross-flow. The amount ofPseudomonas dimunuta contained in the filtrate was measured,demonstrating that it was 10 cells/100 mL or less and the LRV(logarithmic reduction value) of the 0.22 μm indicator bacterium for themodule was 7 or less. From this, it was determined that this moduleenabled almost complete sterilization.

(3) Preparation of Model Fluid for Animal Cell Culture Containing TargetProtein

A serum-free CHO cell culture containing no antibody protein wasprovided which had a salt concentration of about 0.9% by mass (0.15 M),a protein concentration of about 1 g/L and a cell density of 3.0×10⁷/mL,and γ-globulin (from SIGMA) as a target protein was added thereto to aconcentration of 1 g/L to prepare a model fluid for an animal cellculture containing the target protein and being not clarified.

In this model fluid, γ-globulin is a target protein, and all proteinsderived from the cell culture containing a turbid component are impurityproteins.

(4) Analysis of Protein by SDS-PAGE

SDS-PAGE was used to analyze proteins in the culture having passedthrough each of the minimodules prepared in Examples 1, 2 and 6. Thefiltrate (10 μL) to be used for analysis was mixed with an equal amountof a sample treatment solution (Tris SDS sample treatment solution orTris SDSβ ME sample treatment solution from Daiichi Pure Chemical Co.,Ltd.), which was heat-treated at 100° C. for 5 minutes. The resultantsample was applied to a gel plate for electrophoresis (Multigel II Minifrom Daiichi Pure Chemical Co., Ltd.) in an amount of 10 μL per wellusing a micropipette, which was inserted into an electrophoresis tank(EasySeparator™ from Wako Pure Chemical Industries Ltd.) filled with aphoresis buffer (SDS-Tris-glycin phoresis buffer from Daiichi PureChemical Co., Ltd. was used after 1/10 dilution). Phoresis was performedat a constant current of 30 mA for one hour to separate proteins in thefiltrate. The gel plate after phoresis was stained using a stainingreagent (InstantBlue from Funakoshi Corporation or 2D-Silver StainingReagent-II from Daiichi Pure Chemical Co., Ltd.) to confirm proteinbands.

(5) Quantification of HCP

The fluid to be estimated was applied to a 96-well plate in CHO HostCell Protein ELISA Kit from Cygnus Technologies Inc. to quantify HCP asan impurity using Ultrospec Visible Plate Reader II96 from GE HealthcareBioscience Corporation as a plate reader.

(6) Quantification of DNA

The fluid to be estimated was treated using Quant-iT™ dsDNA HS Assay Kitfrom Invitrogen Corporation, followed by quantifying DNA as an impurityemploying Qubit™ fluorometer.

(7) Adsorption Recovery of Target Protein by Protein a Affinity Column

Clear cultures obtained by passage through the minimodules prepared inExamples 1, 2 and 6 were each used as a sample fluid to estimate theadsorption recovery of a target protein therefrom by a protein Aaffinity column. The protein A affinity column (HiTrap Protein A HP 1 mLfrom GE Healthcare Bioscience Corporation) was connected to acommercially available chromatography system (AKTAexplorer100 from GEHealthcare Bioscience Corporation) for fluid-feeding. The buffer forequilibrating the column and washing after sample application used was20 mM sodium-phosphate (pH 7.0), and the buffer for eluting the adsorbedtarget protein used was 0.1 M citric acid-NaOH (pH 3.0). In theadsorption estimation, the following five steps were carried out as onecycle to estimate the adsorption amount of a target protein: 1) pass 10mL of the buffer for equilibration through the column; 2) pass a clearfluid containing the target protein through the column to adsorb thetarget protein thereto; 3) pass 10 mL of a buffer to the column to washimpurity proteins therein; 4) pass 10 mL of the elution buffertherethrough to elute and recover the adsorbed target protein; and 5)pass 10 mL of the buffer through the column to rewash it. At this time,the passage was performed at a flow rate of 1.0 mL/minute in each step.These cycles were continuously performed in the repeated estimation ofthe adsorption recovery of the target protein. The recovered solutionwas diluted by 1/10 and measured for ultraviolet absorbance at 280 nm todetermine the amount of adsorption from the calibration curve obtainedby ultraviolet absorbance measurements at known concentrations inadvance.

(8) Microfiltration Hollow Fiber Membrane Module for Clarification

A minimodule for clarification was prepared by bundling 11 polysulfonatemicrofiltration hollow fibers having an outer diameter of 2.0 mm, aninner diameter of 1.4 mm and a maximum pore size of 0.4 μm and fixingboth ends thereof to a polycarbonate-made module case using an epoxypotting agent so that the hollow parts of the hollow fibers were notblocked. The resultant minimodule had an inner diameter of 0.9 cm, alength of about 8 cm, and an effective membrane area of the hollow fiberinner surface in the module of 39 cm².

(9) Measurement of Dynamic Adsorption Volume

A BSA solution in which BSA was dissolved at a concentration of 1 g/L ina 20 mmol/L Tris-HCl (pH 8.0) buffer was used to pass the BSA solutionthrough the estimation module until the start of its breakthrough. Here,from the concentration (Q) of a BSA solution, the volume (V_(B)) of theBSA solution passed through by the time the breakthrough of theestimation module occurred, and the volume (V_(B)) of an ion-exchangemembrane according to each Example in the estimation module, the dynamicadsorption volume was calculated based on the equation (II) below:

A=Q×V _(B) /V _(M)  (II)

The volume of an ion-exchange membrane is the volume of that from whichthe hollow portion thereof is subtracted. The breakthrough refers to atime point at which the concentration of BSA in the filtrate hasexceeded 0.1 g/L, i.e. 10% of the concentration of the BSA solutionsupplied. The solution was passed from the inside of the hollowion-exchange membrane toward the outside thereof in the estimationmodule. The dynamic adsorption volumes of the anion-exchange membranesprepared in Examples 1, 2 and 6 were 70 mg/mL, 35 mg/mL and 75 mg/mL,respectively, as measured by this method.

Example 1

(i) Introduction of Graft Chains into Porous Hollow Fiber Membrane

In an airtight container was placed porous polyethylene hollow fibershaving an outer diameter of 3.0 mm, an inner diameter of 2.0 mm and amaximum pore size measured by the bubble point method described in theabove (1) of 0.3 μm, and the air in the container was replaced withnitrogen. Then, the hollow fibers were irradiated with 200 kGy of γ-rayto generate radical while cooling the container from the outside thereofwith dry ice. The resultant radical-containing porous polyethylenehollow fibers were placed in a glass reaction tube, and the oxygen inthe reaction tube was removed by depressurization to 200 Pa or less. Areaction solution containing 3 parts by volume of glycidyl methacrylate(GMA) and 97 parts by volume of methanol, adjusted at 40° C., wasinjected thereinto in an amount of 20 parts by mass to the hollow fibersand then allowed to stand in a closed state for 12 minutes to subject tograft polymerization reaction to introduce graft chains into the poroushollow fibers.

The mixed solution was bubbled with nitrogen in advance to replace theoxygen in the mixed solution with nitrogen.

After graft polymerization reaction, the reaction solution in thereaction tube was discarded. The hollow fibers were then washed byplacing dimethylsulfoxide in the reaction tube to remove the residualglycidyl methacrylate, its oligomers and graft chains not fixed to theporous hollow fiber membranes.

After discarding the wash solution, washing was further carried outtwice by placing dimethylsulfoxide therein. Using methanol, washing wassimilarly performed three times. When the hollow fibers after washingwere dried and weighed, the weight of the porous hollow fiber membraneswas 138% of that before introduction of graft chains and the graft ratedefined as the ratio of the graft chain weight to the base materialweight was 38%.

This is equivalent to the ratio of the number of moles of the introducedGMA (molecular weight: 142) to the number of moles of CH₂ groups(molecular weight: 14) as the skeleton unit of the base materialpolyethylene being 3.75% as calculated by the following equation (III).

Mole Number % of Introduced GMA=(Graft Rate/142)/(100/14)×100  (III)

The ratio of the number of moles of the polyethylene skeleton unit CH₂group in the porous hollow fiber membrane after graft reaction and thenumber of moles of ester groups (COO groups) characteristic of GMAconstituting the graft chain was measured by the solid NMR method.

Using 0.5 g of the powdered sample obtained by freezing and grinding thehollow fibers after graft reaction, the measurement was carried out atroom temperature under conditions of a waiting time of 100 s and anintegration of 1,000 times by the quantitative determination mode of theHigh Power Decoupling (HPDEC) method employing DSX400 from BrukerBiospin Corp. and the nuclide ¹³C.

The ratio of the peak area corresponding to the ester group and the peakarea corresponding to the CH₂ group in the resultant NMR spectrum isequivalent to the ratio of the numbers of moles of GMA and CH₂ groups;thus, when the ratio of the number of moles of the introduced GMA to thenumber of moles of CH₂ groups was calculated from the measurementresults, it was found to be 3.8%. This is equivalent to a graft rate of38.5%; it was shown that the graft rate was obtained by measuring thesample after graft reaction by the solid NMR method.

(ii) Fixation of Anion-Exchange Group (Tertiary Amino Group) to GraftChain

A dried hollow fiber into which graft chains were introduced was swollenby immersion in methanol for 10 minutes or more and then immersed inpurified water for replacement with water. The hollow fiber after graftreaction (20 parts by mass) was placed in a glass reaction tubecontaining a reaction solution containing a mixed solution of 50 partsby volume of diethylamine and 50 parts by volume of purified water, andadjusted to 30° C. The porous hollow fiber into which graft chains wereintroduced was inserted thereinto and allowed to stand for 210 minutesto replace the epoxy groups of the graft chains with diethylamino groupsto provide a porous hollow fiber membrane having diethylamino groups asanion-exchange groups.

The resultant porous hollow fiber membrane has an outer diameter of 3.3mm and an inner diameter of 2.1 mm, and 80% of the epoxy groups of thegraft chain in the porous hollow fiber membrane were replaced withdiethylamino groups.

A replacement ratio T was calculated by the following equation (IV),assuming that T is the ratio of the number (N₁) of moles of epoxy groupsreplaced with diethylamino groups to the number (N₂) of moles of epoxygroups.

T=100×N ₁ /N ₂=100×{(w ₂ −w ₁)/M ₁ }/{w ₁(dg/(dg+100))/M ₂}  (IV)

In this equation, M₁ is the molecular weight of diethylammonium (73.14);w₁ is the weight of the porous hollow fiber membrane after graftpolymerization reaction; w₂ is the weight of the porous hollow fibermembrane after replacement reaction with the dimethylamino group; dg isthe graft rate; and M₂ is the molecular weight of GMA (142).

In the same way as described above using the solid NMR method, the ratioof the number of moles of ester groups characteristic of GMA to thenumber of moles of CH₂ groups as polyethylene skeleton units in theporous hollow fiber membrane into which diethylamino groups wereintroduced was measured. As a result, it was found to be 3.75%. This wasequivalent to the graft rate of 38%. From this result, it was determinedthat there was no change in the graft rate due to the introduction ofdiethylamino groups.

(iii) Preparation of Anion-Exchange Membrane Module

A hollow fiber module for the anion-exchange membrane was prepared bybundling three porous hollow fibers having diethylamino groups asanion-exchange groups and fixing both ends thereof to apolysulfonate-made module case using an epoxy potting agent so that thehollow parts of the porous hollow fibers were not blocked.

The resultant module had an inner diameter of 0.9 cm, a length of about3.3 cm, a module inner volume of about 2 mL, an effective volume of theporous hollow fibers in the module of 0.85 mL, and a volume of onlyporous hollow fiber membranes from which hollow parts were subtracted of0.54 mL.

Example 2

(iv) Fixation of Anion-Exchange Groups (Quaternary Amino Groups) toGraft Chain

In the same way as in Example 1, a dried hollow fiber into which graftchains were introduced was swollen by immersion in methanol for 10minutes or more, and then immersed in purified water for replacementwith water. A mixed solution containing 50 parts by volume of purifiedwater and 50 parts by volume of dimethylsulfoxide was provided, andtrimethylammonium chloride was added to a concentration of 0.5M to themixed solution, which was then mixed to provide a uniform reactionsolution. The reaction solution was placed in a glass reaction tube inan amount of 20 parts by mass to the hollow fiber after graft reaction,and adjusted to 60° C. The porous hollow fiber into which graft chainswere introduced was then inserted thereinto and allowed to stand for 200minutes to replace the epoxy groups of the graft chains withtrimethylamino groups to provide a porous hollow fiber membrane havingtrimethylamino groups as anion-exchange groups.

The resultant porous hollow fiber membrane had an outer diameter of 3.2mm and an inner diameter of 2.1 mm, and 80% of the epoxy groups of thegraft chain were replaced with trimethylamino groups. The replacementratio was calculated in the same way as for the diethylamino group bysubstituting the molecular weight of trimethylammonium chloride (95.57)into M₁ in the above equation (IV).

(v) Preparation of Anion-Exchange Membrane Module

A porous hollow fiber membrane module having trimethylamino groups asanion-exchange groups was also prepared in the same way as in Example 1.A module was obtained which has an effective volume of the porous hollowfibers of 0.75 mL and a volume of only porous hollow fiber membranesfrom which hollow parts were subtracted of 0.46 mL.

Example 3

NaCl was added to 20 mM Tris-HCl (pH 8.0) to a concentration of 0.17 Mto prepare a metal salt-containing buffer. In this buffer dissolved wereBSA (pI: 5.6) and γ-globulin at a concentration of 1 g/L each to preparea mixed solution of proteins. This mixed solution of proteins was passedthrough the anion-exchange membrane module having diethylamino groupsprepared in Example 1 at a flow rate of 2 mL/minute, and the filtratewas collected in the form of 5 mL each of fractions. SDS-PAGE was usedto analyze proteins in the fractions having passed through the module.The filtrate (10 μL) to be used for the analysis was mixed with an equalamount of a sample treatment solution (Tris SDS sample treatmentsolution from Daiichi Pure Chemical Co., Ltd.), which was thenheat-treated at 100° C. for 5 minutes. The resultant sample was appliedto a gel plate for electrophoresis (Multigel II Mini from Daiichi PureChemical Co., Ltd.) in an amount of 10 μL per well using a micropipette,which was inserted into an electrophoresis tank (EasySeparator™ fromWako Pure Chemical Industries Ltd.) filled with a phoresis buffer(SDS-Tris-glycin phoresis buffer from Daiichi Pure Chemical Co., Ltd.was used after 1/10 dilution) to perform phoresis at a constant currentof 30 mA for one hour to separate the proteins in the filtrate. The gelplate after phoresis was stained using a staining reagent (InstantBluefrom Funakoshi Corporation).

The results obtained are shown in FIG. 1. Lane 1 is BSA; lane 2 isγ-globulin alone; lane 3 is a mixture liquid of BSA and γ-globulin;lanes 5 to 12 are fractions of the filtrate; and lane 13 is an eluate ofadsorbate. There was the almost exclusive presence of γ-globulin in thefiltrate reaching up to 20 mL (lanes 5 to 8); BSA was totally adsorbed.After adsorption, elution was carried out using a salt solution in which1 M NaCl was dissolved in a buffer. There was the exclusive presence ofBSA in the eluate (lane 13): only BSA was selectively adsorbed to themodule and γ-globulin was non-adsorbed. The results shown in FIG. 1indicated that the method of the present embodiment is effective forseparating a target protein from a salt-containing solution.

Example 4

A serum-free CHO cell culture containing 0.5 g/L of γ-globulin as atarget protein was subjected to dead end filtration using amicrofiltration hollow fiber membrane module to provide a clarifiedsupernatant. This supernatant fluid (54 mL (equivalent to 100 times thehollow fiber membrane volume)) was passed at a flow rate of 2 mL/minutethrough the anion-exchange membrane module having diethylamino groupsprepared in Example 1 to collect all the filtrate. SDS-PAGE was used toestimate proteins in the fractions having passed through the module. Thefiltrate (10 μL) to be used for the analysis was mixed with an equalamount of a sample treatment solution (Tris SDSβ ME sample treatmentsolution from Daiichi Pure Chemical Co., Ltd.), which was thenheat-treated under reduction at 100° C. for 5 minutes. The resultantsample was applied to a gel plate for electrophoresis (Multigel II Minifrom Daiichi Pure Chemical Co., Ltd.) in an amount of 10 μL per wellusing a micropipette, which was inserted into an electrophoresis tank(EasySeparator™ from Wako Pure Chemical Industries Ltd.) filled with aphoresis buffer (SDS-Tris-glycin phoresis buffer from Daiichi PureChemical Co., Ltd. was used after 1/10 dilution). Phoresis was performedat a constant current of 30 mA for one hour to separate proteins in thefiltrate.

The gel plate after phoresis was stained using a staining reagent(2D-Silver Staining Reagent-II from Daiichi Pure Chemical Co., Ltd.).

The results obtained are shown in FIG. 2. Lane 2 is γ-globulin alone;lane 3 is a serum-free CHO cell culture supernatant alone; lanes 4 and 9are serum-free CHO cell culture supernatants containing γ-globulin; andlane 10 is the filtrate for this estimation. It was shown that passagethrough the anion-exchange membrane module removed much impurities inthe salt-containing cell culture to provide a purified target protein.The concentrations of HCP and DNA as typical impurities were 346 μg/mLand 7,200 ng/mL, respectively in the cell culture supernatant beforepassage through the anion-exchange membrane module and considerablydecreased to 39 μg/mL and 52 ng/mL, respectively in the filtrate,indicating that this method was excellent in the property of removingthese protein impurities present in a dissolved state.

Example 5

Using the anion-exchange membrane module containing trimethylaminogroups prepared in Example 2, in the same way as in Example 4, the samesupernatant of the serum-free CHO cell culture containing 0.5 g/L ofγ-globulin as a target protein was passed therethrough in an amount of46 mL equivalent to 100 times the membrane volume to remove impurities.Estimation using SDS-PAGE was performed in the same way as in Example 4.The results are shown as in Example 4 in FIG. 2. Lane 12 is the filtratefor this estimation. It was shown that passage through theanion-exchange membrane module removed much impurities in thesalt-containing cell culture to provide a purified target protein. Theconcentrations of HCP and DNA as typical impurities were 346 μg/mL and7,200 ng/mL, respectively in the cell culture supernatant before passagethrough the anion-exchange membrane module and considerably decreased to73.8 μg/mL and 32.8 ng/mL, respectively in the filtrate, indicating thatthis method was excellent in the property of removing these proteinimpurities present in a dissolved state.

Example 6

The introduction of graft chains and the fixation of diethylamino groupsto the graft chains were carried out in the same way as in Example 1except for the composition of the reaction solution containing 10 partsby volume of glycidyl methacrylate and 90 parts by volume of methanol toprovide a porous hollow fiber membrane containing diethylamino groups asanion-exchange groups and having a graft rate of 140%. As the result ofcalculation using the equation (IV), 93% of the epoxy groups of thegraft chain were replaced by diethylamino groups. The resultant poroushollow fiber membrane had an outer diameter of 4.0 mm and an innerdiameter of 2.5 mm. This porous hollow fiber membrane was used toprepare a hollow fiber module having an effective volume of the poroushollow fibers of 1.53 mL and a volume of only porous hollow fibermembranes from which hollow parts were subtracted of 0.92 mL in the sameway as in Example 1.

Lane 11 in FIG. 2 shows the result of the estimation performed in thesame way as in Example 4 using the hollow fiber module. It was shownthat passage through the module removed many impurities in thesalt-containing cell culture to provide a purified target protein. Theconcentrations of HCP and DNA as typical impurities were 346 μg/mL and7,200 ng/mL, respectively in the cell culture supernatant before passagethrough the anion-exchange membrane module and considerably decreased to42.6 μg/mL and 32.8 ng/mL, respectively in the filtrate, indicating thatthis method is excellent in the property of removing these impuritiespresent in a dissolved state.

Comparative Example 1

Using 3 types of commercially available membranes having anion-exchangegroups, i.e. SartobindQ MA75 (membrane volume: 2.06 mL) from SartoriusAG, MustangQ Acrodisc (membrane volume: 0.18 mL) from Pall Corporation,and BioCap 25 Filter 90ZA (membrane volume: 5 mL or more) from CunoIncorporated, a supernatant of a serum-free CHO cell culture containing0.5 g/L of γ-globulin as a target protein was passed therethrough involumes equivalent to 100 times the respective membrane volumes in thesame way as in Example 4 to remove impurities. Estimation using SDS-PAGEwas performed in the same way as in Example 4. The results are shown asin Example 4 in FIG. 2. Lane 6 was a filtrate of SartobindQ MA75; lane 7was a filtrate of MustangQ Acrodisc'; and lane 8 was a filtrate ofBioCap 25 Filter 90ZA. These results showed that the use of thecommercially available membranes having anion-exchange groups did notallow the effective removal of impurities from the salt-containing cellculture. The concentrations of HCP and DNA in the cell culturesupernatant before passage through the anion-exchange membrane modulewere 346 μg/mL and 7,200 ng/mL, respectively, whereas those after thepassage were 269 μg/mL and 492 ng/mL, respectively, for SartobindQ MA75,248 μg/mL and 3,916 ng/mL, respectively, for MustangQ Acrodisc, and 310μg/mL and 7,600 ng/mL, respectively, for BioCap 25 Filter 90ZA. Theseresults indicated that this method was inferior in the property ofremoving impurities to the method of the present embodiment.

Example 7

Protein A Purification of Solution Having Passed Through DEAAnion-Exchange Membrane

The solution having passed through a hollow fiber membrane module havingdiethylamino groups obtained in Example 4 was used as a cell culturefrom which dissolved impurities were removed to purify γ-globulintherefrom by adsorption and recovery using a protein A affinity column.According to the method described in the above (7), 30 mL of thefiltrate of Example 4 was passed through the protein A affinity columnto adsorb γ-globulin, followed by purification and recovery using anelution buffer. The recovered solution was diluted by 1/10 and measuredfor ultraviolet absorption intensity to estimate the recovery amount. Asa result, 14.2 mg of γ-globulin was contained in the recovered solution;the recovery rate was found to be 95%. The concentrations of HCP and DNAcontained in the recovered solution were 0.162 μg/mL and 11.8 ng/mL,respectively; thus, the method of the present embodiment was shown tohave a high recovery rate and be excellent in the property of removingimpurities.

Example 8

Protein A Purification of Solution Having Passed Through TMAAnion-Exchange Membrane

The solution having passed through a hollow fiber membrane module havingtrimethylamino groups obtained in Example 5 was used as a cell culturefrom which dissolved impurities were removed to purify γ-globulintherefrom by adsorption and recovery using a protein A affinity columnas described in Example 7. According to the method described in theabove (7), 30 mL of the filtrate of Example 5 was passed through theprotein A affinity column to adsorb γ-globulin, followed by purificationand recovery using an elution buffer. The recovered solution was dilutedby 1/10 and measured for ultraviolet absorption intensity to estimatethe recovery amount. As a result, 13.8 mg of γ-globulin was contained inthe recovered solution; the recovery rate was found to be 92%. Theconcentrations of HCP and DNA contained in the recovered solution were0.289 μg/mL and 19.1 ng/mL, respectively; thus, the method of thepresent embodiment was shown to have a high recovery rate and beexcellent in the property of removing impurities.

Example 9

Protein A Purification of Solution Having Passed Through DEAAnion-Exchange Membrane (2)

The solution having passed through a hollow fiber membrane module havingdiethylamino groups obtained in Example 6 was used as a cell culturefrom which dissolved impurities were removed to purify 7-globulintherefrom by adsorption and recovery using a protein A affinity column.According to the method described in the above (7), 30 mL of thefiltrate of Example 6 was passed through the protein A affinity columnto adsorb γ-globulin, followed by purification and recovery using anelution buffer. The recovered solution was diluted by 1/10 and measuredfor ultraviolet absorption intensity to estimate the recovery amount. Asa result, 13.9 mg of γ-globulin was contained in the recovered solution;the recovery rate was found to be 93%. The concentrations of HCP and DNAcontained in the recovered solution were 0.186 μg/mL and 13.8 ng/mL,respectively; thus, the method of the present embodiment was shown tohave a high recovery rate and be excellent in the property of removingimpurities.

Comparative Example 2

Purification was estimated as described in Example 7, using the cellculture containing 0.5 mg/mL of γ-globulin, subjected only toclarification with a microfiltration membrane, obtained in Example 4 asa cell culture from which dissolved impurities were not removed. Thesolution recovered from the protein A affinity column was diluted by1/10 and measured for ultraviolet absorption intensity to estimate therecovery amount. As a result, 14.5 mg of γ-globulin was contained in therecovered solution; the recovery rate was found to be 97%. Theconcentrations of HCP and DNA contained in the recovered solution were2.93 μg/mL and 63.2 ng/mL, respectively. The rate of recovery of thetarget protein was high, but much impurities were contained in therecovered solution in comparison to the case where the impurity removalwas not performed using the hollow fiber membrane module havinganion-exchange groups according to the present embodiment.

Comparative Example 3

The solutions having passed through commercially availableanion-exchange membranes obtained in Comparative Example 1 were used topurify γ-globulin therefrom by adsorption and recovery using a protein Aaffinity column as described in Example 7. The solutions recovered fromthe protein A affinity column were diluted by 1/10 and measured forultraviolet absorption intensity to estimate the rate of recovery fromthe solutions having passed through the respective anion-exchangemembranes. As a result, the recovery rate was high for each of themembranes, i.e. 97% for SartobindQ MA75, 93% for MustangQ Acrodisc, and95% for BioCap 25 Filter 90ZA. The concentrations of HCP and DNA in therecovered solutions were 3.83 μg/mL and 32.6 ng/mL, respectively forSartobindQ MA, 2.54 μg/mL and 46 ng/mL, respectively for MustangQAcrodisc, and 3.27 μg/mL and 88.2 ng/mL, respectively for BioCap 25Filter 90ZA. Thus, this method was inferior in the property of removingimpurities to the method of the present embodiment and only providedalmost the same purification degree as for Comparative Example 2 inwhich no passage treatment using an anion-exchange membrane was carriedout.

The concentrations of HCP and DNA obtained in Examples 4 to 9 andComparative Examples 1 to 3 were shown in Table 1. The results of Table1 showed that the excellent property of removing impurities was obtainedin each of Examples 4 to 9.

TABLE 1 Solution recovered Filtrate from protein A column Treatment HCPDNA HCP DNA method (μg/mL) (ng/mL) (μg/mL) (ng/mL) Examples Diethylamino39 52 0.162 11.8 4 and 7 group module Examples Trimethyl- 73.8 32.80.289 19.1 5 and 8 amino group module Examples Diethylamino 42.6 32.80.186 13.8 6 and 9 group module Comparative Untreated 346 7200 2.93 63.2Example 2 Comparative SartobindQ 269 492 3.83 32.6 Examples MustangQ 2483916 2.54 46 1 and 3 BioCap 90ZA 310 7600 3.27 88.2

Example 10

The cross-flow filtration estimation apparatus shown in FIG. 3 was setup; 300 mL of the cell culture containing a target protein prepared inthe above (3) was placed in a cell culture tank 1 and, using a peristerpump 2, the culture was passed at a linear velocity of 0.5 m/s through ahollow fiber membrane module comprising porous hollow fiber membraneshaving diethylamino groups as anion-exchange groups as prepared inExample 1 (hereinafter referred to as an anion-exchange hollow fibermodule) 4 for cross-flow filtration. Here, internal pressure typefiltration was adopted by which the filtrate moved from the inside ofthe porous hollow fiber toward the outside thereof. The passage pressurewas regulated by a flow control cock 6 so that the average of thepressures measured with a pressure indicator 3 (at the entry side of themodule) and a pressure indicator 5 (at the exit side of the module) was0.1 MPa, and 30 mL each of the solutions having passed through themodule were collected so as to provide a total amount of 210 mL,followed by final collection of 40 mL of the solution and thereby thecross-flow filtration was performed until the total amount of thefiltrate reached 250 mL. During the filtration, the average flux was 21L/m²/hr (21 LMH). All of the filtrates collected were found to be clearby visual observation.

FIG. 4 shows the results of the SDS-PAGE of the filtrates and eluatesperformed according to the method described in the above (4). For all ofthe filtrates collected, the target protein straightly passedtherethrough into the filtrate at the same concentration as that of thestock solution, and it was also shown that impurity proteins wereevidently adsorbed by the anion-exchange hollow fiber membrane module.The anion-exchange hollow fiber membrane module after cross-flowfiltration estimation was back washed with the buffer of 20 mM Tris-HCl(pH 8.0) to remove a turbid component deposited inside the porous hollowfiber, followed by eluting the adsorbed components using a buffersolution containing 1 M NaCl to analyze the eluate by SDS-PAGE. It wasshown that a large amount of impurity proteins were adsorbed to theanion-exchange hollow fiber membrane module, while the target proteinwas not adsorbed to the module.

All of the clear cultures obtained by the passage placed in the samecontainer to make a uniform solution, which was then passed through anunused protein A affinity column to estimate the amount of the targetprotein adsorbed to the protein A affinity column according to themethod described in the above (7). Here, the amount of the culturepassed through the column was 10 mL. In the estimation, the cycle ofadsorption estimation described in the above (7) was repeated 10 timesto measure the change of the adsorption amount. As a result, theadsorption amount in the first cycle was 9.8 mg and the adsorptionamount in the tenth cycle was 9.8 mg, showing no decrease in theadsorption amount during the 10 times repetition. When the eluates werevisually observed, the eluates were found to be clear in all of theestimations and no aggregates were identified. After the end ofestimation, when the protein A affinity column was disassembled toobserve inside beads under an optical microscope, no adhesion ofimpurities to the beads was seen. From these results, it was determinedthat the clarification of a cell culture and the removal of dissolvedimpurity proteins can be simultaneously carried out using theanion-exchange hollow fiber membrane module, which can greatly reduceloads on a protein A affinity column.

Comparative Example 4

Porous polyethylene hollow fibers comprising no anion-exchange groups,having an inner diameter of 3.0 mm and an outer diameter of 2.0 mm wereused to prepare a minimodule by the same method as for theanion-exchange hollow fiber membrane module described in Example 1.Using this minimodule, a filtrate of a cell culture containing a targetprotein was collected in the same way as in Example 3. The resultantfiltrate was clear by visual observation; however, when it was analyzedby SDS-PAGE according to the method described in the above (4), it wasdetermined that an extremely large amount of impurity proteins weredissolved therein compared to the filtrate of Example 10. In addition,when the minimodule after estimation was back washed with a buffer asdescribed in Example 10 and then eluted with a 1 M NaCl buffer solution,no impurity protein or target protein was shown to be adsorbed to theminimodule.

The clear culture obtained by the passage was passed through an unusedprotein A affinity column as described in Example 10 to estimate theadsorption amount of the target protein. When the estimation wasrepeated 10 times without exchanging the column to measure the change ofthe adsorption amount, the adsorption amount in the first estimation was9.8 mg; however, the adsorption amount decreased with increasing numberof repeats, resulting in the adsorption amount in the tenth estimationbeing 9.4 mg. When the eluate was visually observed, white turbidity,although slight, due to aggregates was identified. After the end ofestimation, when the protein A affinity column was disassembled toobserve inside beads under an optical microscope, adhesion of aggregatedimpurities to the beads was seen. This showed that the removal ofdissolved impurity proteins was important for the reduction of loads onthe protein A affinity column, demonstrating the effect of the poroushollow fiber membrane having anion-exchange groups.

Example 11

The cross-flow filtration estimation apparatus shown in FIG. 5 was setup; 300 mL of the cell culture containing a target protein prepared inthe above (3) was placed in a cell culture tank 1 and, using a peristerpump 2, the culture was passed in cross-flow at a linear velocity of 0.5m/s through the microfiltration hollow fiber membrane module 7 forclarification described in the above (8) for filtration. The passagepressure was regulated by a flow control cock 6 so that the average ofthe pressures measured with a pressure indicator 3 (at the entry side ofthe module) and a pressure indicator 5 (at the exit side of the module)was 0.1 MPa. The solution having passed through the microfiltrationhollow fiber membrane module 7 for clarification was directly fed to thesame anion-exchange hollow fiber membrane module 4 as that of Example 10to collect 30 mL each of the filtrates obtained by normal flowfiltration. The time taken until the total filtrate amount reached 250mL was 160 minutes, and the average flux was 120 L/m²/hr (120 LMH). Allof the filtrates collected were found to be clear by visual observation.

When the filtrates collected according to the method described in theabove (4) were analyzed using SDS-PAGE, for all of the filtratescollected, the target protein had straightly passed therethrough intothe filtrate at the same concentration as that of the stock solution,and it was also shown that impurity proteins had been evidently adsorbedby the anion-exchange hollow fiber membrane module.

Example 12

Using the microfiltration hollow fiber membrane module described in theabove (8), the animal cell culture containing a target protein preparedin the above (3) was filtered to provide 300 mL of a clarified (clear)solution. After completely blocking the flow control cock 6 in theestimation apparatus of FIG. 3, the resultant clear solution was placedin the cell culture tank 1 of FIG. 3 and, using the perister pump 2, fedat a feed flow rate of 10 mL/minute therefrom to the same anion-exchangehollow fiber membrane module 4 as that of Example 10 to collect 30 mLeach of the filtrates obtained by normal flow filtration. The time takenuntil the total filtrate amount reached 250 mL was 25 minutes, and theaverage flux was 816 L/m²/hr (816 LMH). All of the filtrates collectedwere found to be clear by visual observation. From these results, it wasdetermined that even the culture containing dissolved impurity proteinscould be subjected to microfiltration before performing the filtrationthereof using the anion-exchange hollow fiber membrane module to removethe impurity proteins at an extremely high processing speed.

When the filtrates collected according to the method described in theabove (4) were analyzed by SDS-PAGE, for all of the filtrates collected,the target protein had straightly passed therethrough into the filtrateat the same concentration as that of the stock solution, and it was alsoshown that impurity proteins had been evidently adsorbed by theanion-exchange hollow fiber membrane module.

As described above, dissolved impurity proteins were capable of beingmarkedly removed in Examples 3 to 12 in which filtration was performedusing the anion-exchange hollow fiber membrane modules comprising poroushollow fiber membranes having anion-exchange groups prepared in Examples1, 2 and 6 compared to in Comparative Examples 1, 3 and 4 in which thepolyethylene hollow fiber membrane modules given no anion-exchangegroups were used, or to in Comparative Example 2 in which noanion-exchange hollow fiber membrane module was used; the solutions ofthe target protein having a higher purity were also capable of beingobtained when purification was performed using affinity chromatography.Particularly in Example 10, even when the solution obtained byperforming the filtration was repeatedly applied to the protein Aaffinity column, the purifying capability of the column was not reduced,the adhesion of impurities to the column was prevented, and loads on thecolumn were sufficiently diminished.

In addition, filtration was performed using the microfiltration hollowfiber membrane module before filtration with the anion-exchange hollowfiber membrane module to enable the filtration step employing theanion-exchange hollow fiber module to be performed at an extremely highprocessing speed. Filtration using the anion-exchange hollow fibermodule could also be performed by normal flow filtration to markedlyremove dissolved impurity proteins.

The present application is based on Japanese Patent Application No.2007-279406 filed Oct. 26, 2007, the content of which is incorporatedherein by reference.

INDUSTRIAL APPLICABILITY

The use of the method for purifying the protein according to the presentembodiment enables the clarification of a cell culture before anaffinity chromatography step, conventionally carried out by the threesteps of centrifugation, microfiltration and sterilization filtration tobe completed by the single step of filtration using a porous hollowfiber membrane having anion-exchange groups. As a result, reduction incost is made possible by simplifying the process and contractingnecessary equipment. Loads in the step of affinity chromatography canalso be considerably reduced to more highly purify a desired protein aswell as to achieve reduced cost of the purification.

1. A method for purifying a protein to remove impurities from a mixture liquid containing a desired protein and the impurities, comprising: performing filtration using a porous membrane having a graft chain on a pore surface and an anion-exchange group fixed to the graft chain.
 2. The method for purifying the protein according to claim 1, wherein the desired protein is one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, and immunoglobulins.
 3. The method for purifying the protein according to claim 1, wherein the impurities are at least one selected from the group consisting of a non-turbid component and a turbid component dispersed in the mixture liquid.
 4. The method for purifying the protein according to claim 3, wherein the non-turbid component is at least one selected from the group consisting of impurity proteins, HCP, DNA, viruses, endotoxins, proteases, and bacteria dissolved in the mixture liquid.
 5. The method for purifying the protein according to claim 3, wherein the turbid component dispersed in the mixture liquid is at least one selected from the group consisting of cells and cell debris.
 6. The method for purifying the protein according to claim 1, wherein a salt concentration of the mixture liquid is from 0.01 M to 0.5 M (both inclusive).
 7. The method for purifying the protein according to claim 6, wherein the salt concentration of the mixture liquid is from 0.1 M to 0.3 M (both inclusive).
 8. The method for purifying the protein according to claim 1, wherein: a base material of the porous membrane is polyethylene or polyvinylidene fluoride, the graft chain is a polymer of glycidyl methacrylate and has a graft rate of from 10% to 250% (both inclusive), and the graft chain has 70% or more of epoxy groups replaced with the anion-exchange groups.
 9. The method for purifying the protein according to claim 8, wherein the graft rate is from 10% to 150% (both inclusive).
 10. The method for purifying the protein according to claim 8, wherein the graft rate is from 10% to 90% (both inclusive).
 11. The method for purifying the protein according to claim 8, wherein the graft rate is from 30% to 60% (both inclusive).
 12. The method for purifying the protein according to claim 1, wherein the anion-exchange group is a diethylamino group and/or a trimethylamino group.
 13. The method for purifying the protein according to claim 1, wherein the anion-exchange group is a diethylamino group.
 14. The method for purifying the protein according to claim 1, wherein the porous membrane has a maximum pore size of from 0.1 μm to 0.8 μm (both inclusive).
 15. The method for purifying the protein according to claim 1, wherein the mixture liquid is filtered using the porous membrane to remove one or more impurities comprising a non-turbid component.
 16. The method for purifying the protein according to claim 1, wherein the mixture liquid is an animal cell culture.
 17. The method for purifying the protein according to claim 1, wherein the porous membrane is a porous hollow fiber membrane.
 18. The method for purifying a protein according to claim 1, further comprising performing purification using affinity chromatography. 